Ceramic/structural protein composites and method of preparation thereof

ABSTRACT

Ceramic/structural protein composites and methods of preparation are disclosed, including coatings and films. Ceramic/structural protein coatings can be fabricated on the surface of substrates, including the surface of implantable medical devices.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 12/265,979filed Nov. 6, 2008, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/985,679 filed Nov. 6, 2007, each of which ishereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has certain rights in this invention pursuant toGrant No. DMI 0500269 awarded by the National Science Foundation.

BACKGROUND OF INVENTION

Implantable medical devices, such as orthopedic and dental prostheses,can be made more permanent if the interface between the existing boneand the device contains some natural bone growth to knit the twocomponents together. Such ingrowth has advantages over the use of bonecement, both in terms of stability and permanency.

“Bioactive” coatings on implantable medical devices allow for theingrowth of natural bone into and around the device, forming chemicalbonds between the device and natural bone. Bone is composed ofsubstituted apatite crystals in an abundant collagen network. Type Icollagen is the major protein of bone tissue, making up about thirtypercent of the weight of bone. It has been shown that apatite crystalscan grow and bond to collagen fibrils, and prepared apatite/collagencomposites have been shown to promote direct bone apposition. However,there are drawbacks to these composites.

Electrophoresis has been used to prepare a bioactive apatite/collagencomposite coating on a substrate. However, this method results in arelatively low bonding strength at the interface between the coating andthe substrate.

Other groups have synthesized apatite/collagen composite coatings by thebiomimetic method using simulated body fluid (“SBF”). The reportedbiomimetic methods took three days to obtain an apatite/collagencomposite coating. The resulting coating contained collagen incolloidal/elliptical particles having sizes over two micrometers. Usinga high saturated SBF solution (concentrated by a factor of five)containing collagen results in an inhomogeneous apatite/collagencomposite coating which is unlike natural bone's ultra-structure at thenano-level. Under these conditions, the collagen fibers in the compositecoating randomly overlapped and submicrometer apatite particles (200-600nm) were attached on the collagen fibers.

Soluble collagen spontaneously forms into gel in neutral salt solutionswithin less than fours hours at physiological temperature (37° C.),while most of the apatite coating starts to deposit on substrate afterfours hours under physiological conditions. As a result undertraditional conditions in the biomimetic process, most of the collagengelates and floats on the surface of the SBF solution or deposits on thesurface of the substrate before the apatite precipitates. In theconcentrated SBF case, apatite deposits on the surface of the collagencoating to form a laminated structure, and not a uniformly mixedcomposite. In both cases, only a small proportion of collagen isincorporated into the apatite coating and forms the nano-levelapatite/collagen composite coating.

There remains a need in the art for improved bioactive compositecoatings in addition to processes to prepare the composite coatings.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, a method of coating a substrate comprises exposing aportion of a substrate to an aqueous system at a temperature of about20° C. to about 80° C. to form a ceramic coating on a surface of thesubstrate; wherein the aqueous system comprises a structural protein, agelation inhibitor agent, a weak acid, water, Ca²⁺, HPO₄ ²⁻, a buffersystem, and optionally one or more of Mg²⁺, Na⁺, K⁺, Cl⁻, SO₄ ²⁻; orHCO₃ ⁻; and wherein the aqueous system has an initial pH of about 5.0 toabout 8.0.

In another embodiment, a method of forming a film comprises forming anaqueous system comprising a structural protein, a gelation inhibitoragent, a weak acid, water, Ca²⁺, HPO₄ ²⁻, a buffer system, andoptionally one or more of Mg²⁺, Na⁺, K⁺, Cl⁻, SO₄ ²⁻; or HCO₃ ⁻; whereinthe aqueous system has an initial pH of about 5.0 to about 8.0; placingthe aqueous system in container allowing for an air-aqueous systeminterface; sealing the container; and allowing a ceramic/structuralprotein film form at the air-aqueous system interface at a temperatureof about 20° C. to about 80° C.

In another embodiment, a device comprises, i) a coated substrateprepared by the process comprising exposing a portion of a substrate toan aqueous system at a temperature of about 20° C. to about 80° C. toform a ceramic coating on a surface of the substrate; wherein theaqueous system comprises a structural protein, a gelation inhibitoragent, a weak acid having a pKa of about 3.0 to about 5.5, water, Ca²⁺,HPO₄ ²⁻, a buffer system, and optionally one or more of Mg²⁺, Na⁺, K⁺,Cl⁻, SO₄ ²⁻; or HCO₃ ⁻; and wherein the aqueous system has an initial pHof about 5.0 to about 8.0; or ii) a film prepared by the processcomprising forming an aqueous system comprising a structural protein, agelation inhibitor agent, a weak acid having a pKa of about 3.0 to about5.5, water, Ca²⁺, HPO₄ ²⁻, a buffer system, and optionally one or moreof Mg²⁺, Na⁺, K⁺, Cl⁻, SO₄ ²⁻; or HCO₃ ⁻; wherein the aqueous system hasan initial pH of about 5.0 to about 8.0; placing the aqueous system incontainer allowing for an air-aqueous system interface; sealing thecontainer; and allowing a ceramic/structural protein film form at theair-aqueous system interface at a temperature of about 20° C. to about80° C.

Also disclosed herein are coatings and films prepared by the processes,as well as uses for the coatings and films.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 a FESEM image of apatite/collagen composite film surfacemorphology at a low magnification.

FIG. 1 b FESEM image of apatite/collagen composite film surfacemorphology at a high magnification.

FIG. 1 c FESEM image of apatite/collagen composite film cross-section ata high magnification.

FIG. 1 d EDX result of apatite/collagen composite film.

FIG. 2 a TEM image of the ultra-thin section of apatite/collagen film

FIG. 2 b TEM image and electron diffraction pattern of apatite/collagencomposite suspension.

FIG. 3 TGA result of the apatite/collagen composite.

FIG. 4 XRD patterns of composite (a) before and (b) after TGA test.

FIG. 5 a FESEM image of composite after TGA test.

FIG. 5 b FESEM image of the composite film after treated with bothglutaraldehyde and EDTA.

DETAILED DESCRIPTION

Disclosed herein are methods of forming ceramic/structural proteincomposite films and coatings; films and coatings prepared therefrom; andarticles prepared therefrom.

The method described herein allows for a mild and convenient approach toform a ceramic/structural protein composite coating, specificallyapatite/collagen composite coating on the surface of a variety ofsubstrates. The method involves immersing a substrate or portion of asubstrate into a coating aqueous system under controlled conditions ofe.g., temperature, pH, ion concentration, and/or buffer to result in theformation of a ceramic/structural protein coating on the substratesurface. When used in implantable medical device applications, theapatite/collagen coating allows for bone ingrowth into the coatingsurface to form a strong bond between the substrate and existing bone.

With the disclosed method, a controllable collagen content denseapatite/collagen composite coating can be formed within twenty-fourhours using a biomimetic method. The biomimetic method results innano-structured, carbonated apatite and collagen composite that ischemically bonded to a substrate through the process of immersing thesubstrate in a coating aqueous system containing calcium, phosphate,structural protein gelation inhibitor agent (e.g., urea can prevent orinhibit collagen gelation) and a structural protein, specificallycollagen. Other ions, such as sodium, potassium, magnesium, chloride,sulfate, and silicate, may optionally be present in the solution alongwith a buffer system.

In one embodiment, a method of coating a substrate with a structuralprotein composite coating comprises exposing at least a portion of asubstrate to a coating aqueous system at a temperature of about 20° C.to about 80° C. to form a composite coating on a surface of thesubstrate; wherein the coating aqueous system comprises structuralprotein, water, Ca²⁺, HPO₄ ²⁻, a weak acid (eg. acetic acid, and thelike), a gelation inhibitor agent (eg. urea, and the like), and a buffersystem; and optionally one or more of the following ions: Mg²⁺, Na⁺, K⁺,Cl⁻, SO₄ ²⁻, HCO₃ ⁻; and wherein the coating aqueous system has aninitial pH of about 50 to about 8.0. By varying the structural proteincontent in the coating aqueous system, the resulting ratio ofceramic/structural protein in the coating can be controlled.

The coating aqueous system generally comprises the following inorganicions: Ca²⁺ and HPO₄ ²⁻, and optionally one or more of the followingions: Mg²⁺, Na⁺, K⁺, Cl⁻, SO₄ ²⁻, and HCO₃ ⁻. The coating aqueous systemcan be prepared by dissolving in an aqueous solvent salts that whendisassociated will result in the particular ions Ca²⁺, Mg²⁺, Na⁺, K⁺,Cl⁻, SO₄ ²⁻, HPO₄ ²⁻ and HCO₃ ⁻. The aqueous solvent can be deionizedand purified water. Exemplary salts include those that result in anaqueous solution of the desired ions, for example, alkali metal halides,alkaline earth metal halides, alkali metal hydrogen carbonates, alkalimetal phosphates, and alkali metal sulfates. Exemplary salts include,NaCl, KCl, K₂HPO₄, MgCl₂, Na₂SO₄, CaCl₂ and NaHCO₃.

The particular concentrations of each of the above-described ionsinitially present in the coating aqueous system can be as follows:

Ca²⁺ at about 2.5 to about 15.0 mM, specifically about 4.0 to about12.0, and more specifically about 8.0 to about 10.0 mM;

Mg²⁺ at about 0 to about 5.0 mM, specifically about 0.5 to about 4.5 mM,and more specifically about 1.5 to about 3.0 mM;

Na⁺ at about 0 to about 300.0 mM, specifically about 50.0 to about 200.0mM, and more specifically about 100.0 to about 150.0 mM;

K⁺ at about 0 to about 20.0 mM, specifically about 2.0 to about 15.0 mM,and more specifically about 7.0 to about 10.0 mM;

Cl⁺ at about 0 to about 350.0 mM, specifically about 50.0 to about 200.0mM, and more specifically about 120.0 to about 150.0 mM;

SO₄ ²⁻ at about 0 to about 2.0 mM, specifically about 0.1 to about 1.0mM, and more specifically about 0.2 to about 0.5 mM;

HPO₄ ²⁻ at about 1.0 to about 10.0 mM, specifically about 3.0 to about8.0 mM, and more specifically about 5.0 to about 7.5 mM; and

HCO₃ ⁻ at about 0 to about 100.0 mM, specifically about 5.0 to about50.0 mM, and more specifically about 20.0 to about 40.0 mM.

An additional component present in the coating aqueous system is abuffer system. The buffer system can contain HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid orN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; Molecular formula:C₈H₁₇N₂SO₃; CAS No: 7365-45-9) and an alkali metal hydrogen carbonate(e.g. NaHCO₃, KHCO3, etc.) which are added to the aqueous system inamounts to substantially stabilize the aqueous system. The concentrationof HEPES present in the aqueous system can be at about 5.0 grams perliter (g/L) to about 80.0 g/L, specifically about 10.0 g/L to about 60.0g/L, and more specifically about 12.0 g/L to about 48.0 g/L.

Additional buffer systems may include tris-hydroxymethyl aminomethan(TRIS), HEPES salts, piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),PIPES salts, combinations of the foregoing with an alkali metalcarbonate, and combinations thereof.

The coating aqueous system may optionally contain additional ioniccomponents such as silicate, strontium, zinc, silver, fluoride,combinations thereof, and the like.

The weak acid present in the aqueous system can be any acid with a pKaof about 3.0 to about 5.5, specifically about 4.5 to about 5.0.Exemplary acids include organic acids, specifically alkyl carboxylicacids such as acetic acid, propionic acid, and the like.

The gelation inhibitor agent is provided in the coating aqueous systemto control the deposition of structural protein (e.g., collagen) suchthat it coincides with ceramic (e.g., apatite) formation. For example,hydrogen bonds between the hydroxyl groups of hydroxyproline and theketo group of the peptide links in the collagen backbone play a majorrole during collagen gelation. To decrease the rate of collagengelation, a quantity of a gelation inhibitor agent such as urea is addedto the aqueous system. By using the gelation inhibitor agent, thedeposition of the structural protein and the apatite onto the substratecan occur simultaneously to obtain a homogenous structuralprotein/apatite nanocomposite coating. For example, when collagen andapatite are deposited in the presence of urea, a nano-composite coatingis formed having a structure similar to natural bone.

The coating aqueous system used to prepare the coatings can contain anamount of a gelation inhibitor agent to allow for the controlleddeposition of structural protein and apatite onto a substrate. Exemplarygelation inhibitor agents include urea, histidine, hydroxyproline,thiourea, sodium dodecyl sulfate, lithium dodecyl sulfate,2-mercaptoethanol, formamide, dithiothreitol, CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), CHAPSO(3-([cholamidopropyl]-dimethyl ammonio)-2-hydroxy-1-propanesulfonate),guanidinium chloride (guanidine hydrochloride), guanidinium thiocyanate,and the like; specifically urea. The amount of gelation inhibitor agentpresent in the coating aqueous system can be about 0.1 M to about 6.0 M,specifically about 0.2 M to about 1.0 M, and more specifically about 0.4M to about 0.6 M.

The structural protein used in the structural protein containing aqueoussystem can be any known structural protein such as collagens, elastin,and keratins, specifically collagen, and more specifically solublecollagen Types I, II, III, and V, and yet more specifically collagenType I. As used herein, soluble collagen means “collagen molecules ormicrofibrils which are soluble in an aqueous solution”.

There is no particular limitation as to the source of the structuralprotein. The structural protein may be obtained from commercial sourcesor extracted from natural sources using procedures well known in theart.

In one embodiment, a collagen containing coating aqueous system containsabout 0.01 to about 0.9 g/L collagen solution, about 5.0 to about 10.0mM Ca²⁺, about 1.0 to about 5.0 mM HPO₄ ²⁻, about 100.0 to about 200.0mM Na⁺, about 2.0 to about 8.0 mM K⁺, 1.0 to about 2.0 mM Mg²⁺, 50.0 toabout 150.0 mM Cl⁻, about 5.0 to about 50.0 mM HCO₃ ⁻, about 1 to about1.0 mM SO₄ ²⁻, about 0.3 to about 0.7 M urea, about 0.1 to about 0.3Macetic acid, and about 5.0 to about 15.0 g/L HEPES.

The density/porosity of the ceramic coating can be adjusted by severalparameters including amount of HCO₃ ⁻, initial pH of the coating aqueoussystem, amount of buffer, temperature of the coating process, calciumconcentration, phosphate concentration and other ion concentrations(i.e. Mg²⁺).

The density/porosity of the ceramic coating can be adjusted by carefullychoosing the initial pH of the coating aqueous system. Over time, the pHof the aqueous system increases due to the bicarbonate ions in thesolution naturally decomposing into hydroxyl groups and carbon dioxide.The initial formation of the coating is formed when the aqueous systemhas an initial pH of about 5.0 to about 8.0. The initial stage of thecoating process is slower as HCO₃ ⁻ inhibits the crystal growth of thecoating. Therefore, the coating will grow slower and denser at theinitial stages of the coating process as the concentration HCO₃ ⁻ isinitially high. As the HCO₃ ⁻ ions decompose, the rate of coatingformation increases and the inhibitory effect of the bicarbonate ions isless pronounced.

The amount of buffer in the aqueous system will also alter the pH changeprofile during the coating process. When there is less buffer in theaqueous system, more HCO₃ ⁻ will be present in the system when the pHrange for apatite formation is achieved.

The calcium and phosphate concentrations can also be chosen to obtainthe optimal pH range for apatite formation. Magnesium ions are known todecrease the rate of apatite formation and thereby attributes to theformation of a relatively dense coating.

If needed, the initial pH of the coating aqueous system can be adjustedby the addition of an inorganic acid or inorganic base. An exemplaryinorganic acid includes halo acids (e.g. hydrochloric acid). Exemplaryinorganic bases include alkali metal hydroxides (e.g. NaOH, KOH, etc.).The initial pH of the aqueous system can be about 5.0 to about 8.0,specifically about 5.8 to about 7.5, more specifically about 6.0 toabout 6.60, yet more specifically about 6.10 to about 6.45, and stillyet more specifically about 6.20 to about 6.38. As used herein, “initialpH” means the pH of the coating aqueous system prior to contact with thesubstrate to be coated.

The initial pH of the coating aqueous system and the type and amount ofbuffer system can be selected to generate a desired ceramic coating.After the desired coating aqueous system is prepared, the substrate isexposed to the coating aqueous system at a particular temperature toallow for the formation of the coating. The substrate can be exposed tothe coating aqueous system for a time sufficient for the formation of acoating of sufficient thickness. Coatings having sufficient thicknesscan be formed in less than about 3 days. Specifically, the substrate canbe exposed in the aqueous system for about 1 to about 48 hours,specifically about 10 to about 40 hours, more specifically about 12 toabout 35 hours, and yet more specifically about 20 to about 30 hoursuntil the desired thickness of coating is formed.

In a generalized process, substrates to be coated are placed in a 2Xvolume container containing X volume of coating aqueous system. Thecontainer is sealed and coating formation is allowed to proceed forabout twenty-four hours at about 40° C. After immersion the substrateswere removed, gently washed with de-ionized water and air-dried.

The process allows for the variation of structural protein content thatis incorporated into the ceramic/structural protein composite coating.Amounts of structural protein (e.g. collagen) incorporated in thecoating can be about 0.01 to about 30 weight percent, specifically about5 to about 25 weight percent, more specifically about 10 to about 20weight percent, and yet more specifically about 13 to about 17 weightpercent based on the total weight of the coating.

Apatite/collagen composite coatings have been shown to be more effectivethan apatite coating in improving cell attachment and activity ofosteoblast-like cells. In the present methods, the amount of collagencontent in the apatite/collagen composite coatings can be optimizedthrough the ability to control the collagen weight percent in thecoating. Furthermore, the bonding strength of the apatite/collagencomposite coating to the substrate is high.

The bonding strength of the coating can be determined using a modifiedASTM C-633 method as provided in Kim H-M, Miyaji F, Kokubo T, NakamuraT. “Bonding strength of bonelike apatite layer to Ti metal substrate.”Journal of Biomedical Materials Research 1997; 38(2):121-127, which isincorporated herein in its entirety.

The coating methods are performed at low temperatures suitable fortemperature sensitive substrates such as polymeric materials andhydrogels. The coating process can be performed at a relatively shortamount of time. Furthermore, the methods can be used to coat poroussubstrates and substrates having complex geometries. Additionalembodiments are directed to the ceramic coatings themselves as well asarticles prepared from substrates comprising the ceramic coatings. Ingeneral, the ceramic/structural protein coating can be prepared byexposing a portion of a substrate to a coating aqueous system comprisinginorganic ions and a structural protein such as Type I soluble collagen.The substrate is exposed for a period of time and at a temperature toallow for the formation of the ceramic/structural protein coating on theexposed surface of the substrate. Exposing can include immersion of thesubstrate or portion of the substrate to the coating aqueous system. Theresulting ceramic coating is generally a bone-like apatite, but can alsobe other types of calcium phosphate. Exemplary calcium phosphateminerals include Ca₅(PO₄)_(3-x)(OH)_(1-y)(CO₃)_(x+y), Ca₅(PO₄)₃(OH),Ca₃(PO₄)₂, CaHPO₄, Ca(H₂PO₄)₂, and the like.

As used herein “exposing a portion of a substrate” means any portion orall of the substrate is exposed to the aqueous system.

The temperature of the coating aqueous system during the coating processcan be about 20 to about 100° C., more specifically about 25 to about80° C., yet more specifically about 35 to about 60° C., and still yetmore specifically about 38 to about 45° C. In one embodiment, thetemperature of the coating aqueous system can be varied during thecoating process. At different temperatures, the optimal pH range forapatite formation will also be different as the rate of HCO₃ ⁻decomposition is affected by temperature. By increasing the temperature,the greater the rate of HCO₃ ⁻ decomposition as compared to lowertemperatures for same time period.

In another embodiment, the temperature of the collagen containingcoating aqueous system during the coating process can be about 20 toabout 50° C., the initial pH of about 5.5 to about 8.0, the collagen atabout 0.1 g/L to about 5.0 g/L, the urea at about 0.1 M to about 6.0 M,the HCO₃ ⁻ at about 10 to about 150 mM, HPO₄ ²⁻ at about 1 to about 10mM, Ca²⁺ at about 2.5 to about 15 mM, and HEPES at about 5 g/L to about80 g/L.

In yet another embodiment, the temperature of the collagen containingcoating aqueous system during the coating process can be about 25 toabout 50° C., the initial pH is about 5.5 to about 8.0, the collagen atabout 0.3 g/L to about 3.0 g/L, the urea at about 0.2 M to about 3.0 M,HCO₃ ⁻ at about 20 to about 100 mM, HPO₄ ²⁻ at about 3 to about 8 mM,Ca²⁺ at about 4 to about 13 mM, and HEPES at about 10 g/L to about 50g/L.

In yet another embodiment, the temperature of the collagen containingcoating aqueous system during the coating process can be about 35 toabout 45° C., the initial pH is about 6.38 to about 6.45, the collagenat about 0.5 g/L to about 1.5 g/L, the urea at about 0.3 M to about 1.0M, HCO₃ ⁻ at about 30 to about 40 mM, HPO₄ ²⁻ at about 2.5 to about 3.5mM, Ca²⁺ at about 7 to about 9 mM, and HEPES at about 10 g/L to about 14g/L.

In still yet another embodiment, the temperature of the collagencontaining coating aqueous system during the coating process can beabout 35 to about 45° C., the initial pH is about 6.00 to about 6.10,the collagen at about 0.7 g/L to about 1.0 g/L, the urea at about 0.4 Mto about 0.5 M, HCO₃ ⁻ at about 60 to about 80 mM, HPO₄ ²⁻ at about 4.5to about 5.5 mM, Ca²⁺ at about 11 to about 13 mM, and HEPES at about 42g/L to about 45 g/L.

Generally, the longer the substrate is exposed to the coating aqueoussystem, the thicker the resulting composite coating will be. Coatingshaving a total thickness of about 0.1 to about 70 micrometers can beformed, specifically about 1 to about 50 micrometers, yet morespecifically about 5 to about 40 micrometers, and still yet morespecifically about 10 to about 25 micrometers.

Exemplary substrates that can be coated with the described compositecoating include implantable medical devices useful in biomedicalapplications, including orthopedic applications (e.g., joint prostheses)and devices and appliances for orthodontic applications and dentalimplants. The aqueous system lends itself to the uniform application ofa ceramic coating even to substrates having surfaces of complexgeometries. Additional applications in the biomedical field includedrug/protein delivery devices. In addition, this coating system can alsobe used to load living cells, coat the surface of tissue engineeringscaffold and other soft tissue replacement materials.

The coatings can be used to prepare medical, surgical, reconstructive,orthopedic, orthodontic, prosthodontic, endodontic or dental devices,implants, appliances, or a component thereof (e.g., a screw or otherattaching connector, etc.).

The substrates can be made from a wide variety of material types,including metal, ceramic, polymeric materials, silicon, glass, and thelike. When used in biomedical applications, the material should bebiocompatible. As used herein, “biocompatible” means being biologicallycompatible in that a toxic, injurious, or immunological response is notproduced in living tissue. Suitable material for the substrate includes,for example, titanium, stainless steel, nickel, cobalt, niobium,molybdenum, zirconium, tantalum, chromium, alloys thereof andcombinations thereof. Exemplary polymeric material include polylactide(PLA), poly(glycolic acid) (PGA), poly(methyl methacrylate) (PMMA),other biocompatible polymeric material, and the like. Exemplary ceramicmaterials include alumina, titania, and zirconia, glasses, and calciumphosphates, such as hydroxyapatite and tricalcium phosphate.

Prior to the coating step, the surface of the substrate can be preparedto improve the adhesion of the coating. The substrate can be cleaned ortreated to remove any surface contaminants. The metal substrates can besurface treated by sand-blasting, scoring, polishing, and grinding toincrease the surface roughness. Alternatively, the metal substrate canundergo chemical surface treatments prior to coating to provide a levelof surface roughness. Exemplary chemical treatments for metal substratesinclude, acid etchings with strong mineral acids, such as hydrofluoric,hydrochloric, sulfuric, nitric and perchloric acids; treatment withstrong alkalis, such as sodium hydroxide, potassium hydroxide; treatmentwith oxidizing agents such as peroxyhalogen acids, hydroxyperoxides, orhydrogen peroxide to form a metal oxide layer. Washing with deionized orpurified water can effect removal of surface contaminants due to thesurface treatment.

In another embodiment, a method of coating a substrate with aceramic/structural protein coating comprises exposing a portion of asubstrate to a collagen containing coating aqueous system in a closedsystem, e.g., a sealed container, at a temperature of about 20° C. toabout 45° C. to form a ceramic/collagen coating on a surface of thesubstrate, wherein the collagen containing coating aqueous systemcomprises water, collagen, urea, Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl⁻, HPO₄ ²⁻, HCO₃⁻ and a buffer system, wherein the coating aqueous system has an initialpH of about 5.5 to about 8.0, and wherein the closed system comprises avolume ratio of headspace to aqueous system of about 5 to about 15 atatmospheric pressure.

Although the coatings have been discussed in terms of its applicationfor implantable medical devices, the coatings can be used for a widevariety of uses, such as a drug delivery carrier, for example. Thecoatings can be useful in hard tissue replacement materials and alsosoft tissue replacement materials.

As used herein “bioactive” means the ceramic coating can induce boneingrowth resulting in the formation of a strong bond across theinterface between the coating and the natural bone.

In one embodiment, a reactor vessel is used in the coating process. Thereactor vessel comprises a liquid-holding container and a gas valve(“pressure valve”) to control the rate of release of a gas from thecontainer. The liquid-holding container can be prepared from anon-reactive or inert material such as glass or Teflon™.

The shape of the interior of the liquid-holding container can be aregular shape such as a cube, a cone, a sphere, a cylinder, or the like.Optionally, the shape of the interior of the liquid-holding containercan be similar to the shape of the substrate to be coated. In order tohave the container with a shape similar to the substrate, the containercan be molded to follow the shape of the substrate to be coated.

In one embodiment, the shape of the interior of the liquid-holdingcontainer is hemispheric and the substrate is a hip acetabular cap.

The liquid-holding container can optionally have a volume sufficient toallow a ratio of the coating aqueous system volume to the substratesurface area to be about 5 to about 50.

The gas valve is a gas-releasing valve used to control the rate ofrelease of a gas, such as carbon dioxide, from the liquid-holdingcontainer. The gas valve can be a manual gas valve, apressure-responsive gas valve, or an automated gas valve.

Also disclosed herein is a convenient method of preparing denseceramic/structural protein composite films at the air-solution interfaceof a structural protein (e.g., soluble collagen) containing concentratedaqueous system. The coating aqueous systems for preparing theceramic/structural protein composite coatings discussed above can beused to prepare the composite films. Furthermore, similar conditions forthe coating process above, with regard to time and temperature, can beused for preparing the apatite/collagen composite film.

The apatite/collagen composite film can be isolated and optionallypurified (e.g., by washing with deionized/purified water). As the filmhomogenously contains structural protein and hydroxyapatite particles,it can be prepared into a powder and used as a filler material orcompressed into a variety of shapes for bone repair/fixation. Theresulting material will have mimicking properties of allograftmaterials. Any process known in the art to convert the film to powder(e.g., grinding and the like) can be used.

EXAMPLES Example 1 Apatite/Collagen Composite Coating: Biomimetic Method

Type I collagen was extracted from rat tail tendon as previouslydescribed in W. Zhang, S. S. Liao, F. Z. Cui, Chem. Mater. 2003, 15,3221. The rat tail tendon was soaked in 0.5 M acetic acid for 3-4 daysat 4° C. The solution was centrifuged at 10,000 rpm at 4° C. for 15minutes and filtered with No. 1 filter paper to remove the insolublecomponents. NaCl (5% wt %) was added to induce precipitation ofcollagen, and the precipitates were collected by centrifuging at 10,000rpm for 15 minutes at 4° C. A collagen solution of ˜1.5 grams per liter(g/L) was formed. From this solution, dilute collagen solutions wereprepared into different concentrations as shown in Table 1. Urea with aconcentration of 0.5 M was added to the collagen solution.

A collagen-containing aqueous system was prepared containing 7.5 mM Ca²⁺and 3.0 mM HPO₄ ²⁻, 142.0 mM Na⁺, 5.0 mM K⁺, 1.5 Mg²⁺, 103.0 mM Cl⁻,27.0 mM HCO₃ ⁻, 0.5 mM SO₄ ²⁻, 0.5M Urea, and 0.2M acetic acid; preparedfrom NaCl, NaHCO₃, Na₂CO₃, KCl, K₂HPO₄.3H₂O, MgCl₂.H₂O,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (11.928 g per1000 mL water), CaCl₂, Na₂SO₄, Urea, glacial acid, and 5M NaOH (6.5 mLper 1000 mL water). Analytical grade reagents NaCl, NaHCO₃, MgCl₂,K₂HPO₄ and CaCl₂ were dissolved into de-ionized water with desiredamounts. HEPES was chosen to buffer the solution and the initial pH wasadjusted to about 6.00 to about 7.00 using 5M NaOH.

A commercially available titanium plate (McMaster-Carr) was cut into15×15×1 millimeter (mm) plates. The titanium plates were polished usinga series of silicon carbide papers (grade 600-1200), and then rinsedwith de-ionized water in an ultrasonic bath. The titanium plates weredried at room temperature overnight. The clean titanium alloy plateswere then soaked in 5 M NaOH solution at 60° C. for 1 day. Afteralkaline treatment, the titanium plates were gently cleaned withde-ionized water. The titanium plates were immersed in 50 milliliters(ml) of the collagen-containing aqueous system in a sealed 100 ml bottleand allowed to form apatite coating. The coating formation process wasstudied at 40° C. After immersion for 24 hours, the plates were removedfrom each solution, gently washed with de-ionized water and air-driedfor overnight.

The resulting composite coatings were then characterized withthermogravimetric analysis (TGA). The analyses revealed a controllablecollagen content composite coating was achieved.

TABLE 1 Coating A Coating B Coating C Collagen in aqueous 1.5 g/L 0.9g/L 0.45 g/L solution Collagen in composite 18% 11% 7% coating (wt %)

Example 2 Apatite/Collagen Composite: Biomimetic Method

Type I collagen was extracted from rat tail tendon as previouslydescribed W. Zhang, S. S. Liao, F. Z. Cui, Chem. Mater. 2003, 15, 3221.The rat tail tendon was soaked in 0.5 M acetic acid for 3-4 days at 4°C. The solution was centrifuged at 10,000 rpm at 4° C. for 15 minutesand filtered with No. 1 filter paper to remove the insoluble components.NaCl (5% wt %) was added to induce precipitation of collagen, and theprecipitates were collected by centrifuging at 10,000 rpm for 15 minutesat 4° C. Collagen was then dissolved in 0.5 M acetic acid to form acollagen solution with a concentration of 0.5 mg/ml. Urea with aconcentration of 0.5 M was added to the collagen solution. Analyticalgrade reagents NaCl, NaHCO₃, MgCl₂, K₂HPO₄ and CaCl₂ with desiredamounts were placed into the collagen solution. HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was chosen tobuffer the so formed collagen-containing aqueous system. The ionconcentrations of the aqueous system are listed in Table 2.

TABLE 2 Collagen containing-aqueous Ion system (concentration in mM) Na⁺109.5 K⁺ 6.0 Mg²⁺ 1.5 Ca²⁺ 7.5 Cl⁻ 110.0 HCO₃ ⁻ 17.5 HPO₄ ²⁻ 3.0 SO₄ ²⁻0 Urea 500 Collagen 0.5 g/LThe collagen-containing aqueous system was stirred for 10 minutes, andits pH was then adjusted to 6.2 using 5 M sodium hydroxide at roomtemperature.

Collagen-containing aqueous system of 50 ml was added to a sealed 100 mlbottle to prepare apatite/collagen composite. The composite formationwas taken place at 40° C. After the solution was aged for 24 hours, thecomposite was formed at the air/solution interface. The composite wasremoved from the solution, gently washed with de-ionized water andair-dried.

The morphology of the composite film was analyzed using a field emissionscanning electron microscope (FESEM) (JEOL JSM 6335F). The specimenswere coated with gold before FESEM observations. An FESEM image of thecomposite is shown in FIG. 1 a. A higher magnification view (FIG. 1 b)shows that the composite is very dense with minimized number of pores atsizes smaller than 100 nanometer (nm). The cross-section morphology ofthe composite (FIG. 1 c) also suggests that the composite is extremelydense, and no pore is observed.

Energy dispersive X-ray (EDX) microanalysis (FIG. 1 d) reveals that thecomposite film is compose of four elements: calcium (Ca), phosphorous(P), oxygen (O), and carbon (C).

The microstructure of apatite/collagen composite film was studied usingtransmission electron microscopy (TEM). TEM samples were prepared bysuspending the composite in an ethanol solution and sonicated for 40minutes to break up the specimen. A drop of the suspension was thenplaced onto a copper electron microscope grid. After air-dried, thesample was observed using a JEOL JEM-2010 TEM at 120 kV. The ultra-thinsection of the composite was also observed using TEM (FEI Tecnai G²Biotwin TEM) at 80 kV. The composite film was embedded in Spurr lowviscosity epoxy resin (Electron Microscopy Sciences). Ultra-thinsections of the composite were prepared using LKB Ultrotome Vultramicrotomes with diatome diamond knifes, and the sections weretransferred onto copper electron microscope grids for TEM observation.

A TEM image (FIG. 2 a) shows that the apatite/collagen composite formsinto plate-like structure with dimensions around 100 nm×100 nm withthickness about 5 nm (dark line is the cross-section of the plate). FIG.2 b shows the TEM image of nano-sized (around 5 nm in diameter) apatiteparticles within the composite plate. A typical selected area electrondiffraction (SAED) pattern of the composite is also shown in FIG. 2 b,where all the rings are attributed to hydroxyapatite (PDF 9-432).

The collagen weight percentage was determined using TGA (TA InstrumentsTGA Q-500) measurement. The film was heated from room temperature to800° C. at 10° C./min.

Thermal gravimetric analysis (TGA) profile (FIG. 3) implies that theweight loss of the composite can be divided into three stages.Approximately 6 wt % weight loss occurred at both below 250° C. andwithin 250-600° C., but only 0.5 wt % weight loss was observed in thetemperature range 600 to 800° C. Previous studies suggested that theweight loss in the first stage was mainly associated with water loss,while that in the second stage was attributed to the loss of the organicmaterial (collagen), and then the decomposition of carbonated apatiteled to the weight loss in the third stage. The finial remaining productshould be pure calcium phosphate. The TGA result revealed thatapproximately 6 wt % of the composite was made of organic material(collagen).

The as-prepared and heat-treated (after TGA test) composites weregrinded into powder and examined using X-ray diffractometer (BRUKER AXSD5005) with a copper target. The voltage and current setup were 40 kVand 40 mA, respectively. A step size of 0.02° and a scan speed of0.5°/min were used.

The XRD result (FIG. 4 a) of the as-prepared composite shows that twobroad peaks are present at 26° and 32°. These peaks indicated thatnano-sized apatite particles were formed in the composite. This resultwas consistent with the TEM observation that the apatite particlesformed in the composite were in the nanometer range. After the TGA test,both apatite and β-tricalcium phosphate (TCP) co-existed in thecomposite (FIG. 4 b), which suggested that the apatite in the compositehad a low decomposition temperature (<800° C.) compared with thoseprepared by a wet process with a decomposition temperature between1250-1450° C.

The morphology of the heat treated (after TGA test) composite films werealso evaluated using FESEM. The specimens were coated with gold beforeFESEM observations. After calcination, all collagen in the composite isburned out leaving pure calcium phosphates. The FESEM image (FIG. 5 a)reveals that apatite particles with an average size around 100 nm areclosely packed together. The pores among the apatite particles areinterconnected with a diameter 100-200 nm. The apatite particles (˜100nm, FIG. 5 a) after the TGA test are much larger than those of asprepared (˜5 nm, FIG. 2 b) due to the growth of apatite particles athigh temperatures. The morphologies of the specimens before and afterthe TGA test were analyzed. Not wishing to be bound by theory, it isbelieved that the collagen was filled in the voids among apatiteparticles in the composite before the TGA test.

After soaking in 3% glutaraldehyde solution for 1 hour to crosslink thecollagen within the composite, the composite was rinsed with de-ionizedwater for several times to completely remove glutaraldehyde residues.The composite was then soaked in 0.25 M ethylenediaminetetraacetate(EDTA) solution for 2 hours to dissolve apatite in the composite. Theremaining material was carefully collected, rinsed with de-ionized waterfor several times and air-dried. The specimen was subsequently coatedwith gold for FESEM observation. The elements in both the as-preparedcomposite and the remaining material after glutaraldehyde and EDTAtreatments were determined using an environmental scanning electronmicroscopy (ESEM) (ESEM 2020 Philips) equipped with energy-dispersivex-ray (EDX) using an EDAX CDU leap detector system.

After treated with glutaraldehyde and EDTA, the collagen in thecomposite was cross-linked and fixed before the calcium phosphate wasremoved by EDTA. EDX examination of the treated sample (FIG. 5 b)exhibited that only copper (Cu), carbon (C), oxygen (O) and sulfur (S)were present in the specimen, where the copper signal came from thesupporting TEM copper grid. The carbon, oxygen and sulfur peakssuggested that only protein remained in the composite after thetreatments as it was expected. Compared with the EDX result of theas-prepared composite, it was noted that both calcium and phosphateelements existed in the composite before glutaraldehyde and EDTAtreatments. It was also revealed by the FESEM observation (FIG. 5 b)that the remaining collagen demonstrated irregular flake shape withdimensions of 200-500 nm×˜200 nm×˜20 nm.

As shown by this Example, aggregation of apatite nanoparticles andcollagen molecules occur simultaneously at the air/water interface todevelop an apatite/collagen composite. In the collagen-containingaqueous system, both collagen and apatite could self-assemble into smallnuclei when the pH of the solution reached a certain range. Based on theapatite morphology after the TGA test, it is inferred that the apatiteparticles are evenly distributed among the collagen fibrils. Thesecollagen fibrils and apatite nanoparticles demonstrated a flake-likestructure, which then formed into dense apatite/collagen nanocomposites.The apatite/collagen composite formed into a platelet shape with onehundred nanometers in each dimension and less than 10 nm in thickness.The nano-sized apatite (˜5 nm) particles were evenly distributed withinthe composite. These apatite/collagen composite platelets were stronglyadhered to each other and formed into dense composite films.

The terms “a” and “an” herein do not denote a limitation of quantity,but rather denote the presence of at least one of the referenced item.The suffix “(s)” as used herein is intended to include both the singularand the plural of the term that it modifies, thereby including one ormore of that term (e.g., the metal(s) includes one or more metals).Ranges disclosed herein are inclusive and independently combinable(e.g., ranges of “up to about 25 wt %, or, more specifically, about 5 wt% to about 20 wt %”, is inclusive of the endpoints and all intermediatevalues of the ranges of “about 5 wt % to about 25 wt %,” etc).

While the invention has been described with reference to a preferredembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing fromessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

1. A method of forming a film, comprising: forming an aqueous systemcomprising a structural protein, a gelation inhibitor agent, a weak acidhaving a pKa of about 3.0 to about 5.5, water, Ca²⁺, HPO₄ ²⁻, a buffersystem, and optionally one or more of Mg²⁺, Na⁺, K⁺, Cl⁻, SO₄ ²⁻; orHCO₃ ⁻; wherein the aqueous system has an initial pH of about 5.0 toabout 8.0; placing the aqueous system in a container and allowing for anair-aqueous system interface; sealing the container; and allowing aceramic/structural protein film form at the air-aqueous system interfaceat a temperature of about 20° C. to about 80° C.
 2. The method of claim1, wherein the structural protein is collagen Type I, II, III, or V. 3.The method of claim 1, wherein the gelation inhibitor agent is urea,histidine, hydroxyproline, thiourea, sodium dodecyl sulfate, lithiumdodecyl sulfate, 2-mercaptoethanol, formamide, dithiothreitol,(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate),(3-([cholamidopropyl]-dimethyl ammonio)-2-hydroxy-1-propanesulfonate),guanidinium chloride (guanidine hydrochloride), or guanidiniumthiocyanate.
 4. The method of claim 1, wherein the buffer systemcomprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid salts,tris-hydroxymethyl aminomethan, piperazine-1,4-bis(2-ethanesulfonicacid), piperazine-1,4-bis(2-ethanesulfonic acid) salts, or combinationsthereof.
 5. The method of claim 1, wherein the weak acid is an organiccarboxylic acid having a pKa of about 3.0 to about 5.5.
 6. A filmprepared by the process of claim
 1. 7. A device, comprising: i) a coatedsubstrate prepared by the process comprising exposing a portion of asubstrate to an aqueous system at a temperature of about 20° C. to about80° C. to form a ceramic coating on a surface of the substrate; whereinthe aqueous system comprises a structural protein, a gelation inhibitoragent, a weak acid having a pKa of about 3.0 to about 5.5, water, Ca²⁺,HPO₄ ²⁻, a buffer system, and optionally one or more of Mg²⁺, Na⁺, K⁺,Cl⁻, SO₄ ²⁻; or HCO₃ ⁻; and wherein the aqueous system has an initial pHof about 5 to about 8; or ii) a film prepared by the process comprisingforming an aqueous system comprising a structural protein, a gelationinhibitor agent, a weak acid having a pKa of about 3.0 to about 5.5,water, Ca⁺, HPO₄ ²⁻, a buffer system, and optionally one or more ofMg²⁺, Na⁺, K⁺, Cl⁻, SO₄ ²⁻; or HCO₃ ⁻; wherein the aqueous system has aninitial pH of about 5 to about 8; placing the aqueous system in acontainer and allowing for an air-aqueous system interface; sealing thecontainer; and allowing a ceramic/structural protein film form at theair-aqueous system interface at a temperature of about 20° C. to about80° C.
 8. The device of claim 7, wherein the structural protein iscollagen Type I, II, III, or V.
 9. The device of claim 7, wherein thegelation inhibitor agent is urea, histidine, hydroxyproline, thiourea,sodium dodecyl sulfate, lithium dodecyl sulfate, 2-mercaptoethanol,formamide, dithiothreitol,(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate),(3-([cholamidopropyl]-dimethyl ammonio)-2-hydroxy-1-propanesulfonate),guanidinium chloride (guanidine hydrochloride), or guanidiniumthiocyanate.
 10. The device of claim 7, wherein the buffer systemcomprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid salts,tris-hydroxymethyl aminomethan, piperazine-1,4-bis(2-ethanesulfonicacid), piperazine-1,4-bis(2-ethanesulfonic acid) salts, or combinationsthereof.
 11. The device of claim 7, wherein the device comprises thecoated substrate; the structural protein is collagen Type I present inan amount of about 0.1 g/L to about 5.0 g/L; the gelation inhibitoragent is urea present in an amount of about 0.1 M to about 6.0 M; Ca²⁺is present in an amount of about 2.5 to about 15.0 mM; Mg²⁺ is presentin an amount of about 0.5 to about 5.0 mM; Na⁺ is present in an amountof about 50.0 to about 300.0 mM; K⁺ is present in an amount of about 2.0to about 20.0 mM; Cl⁻ is present in an amount of about 50.0 to about350.0 mM; SO₄ ²⁻ is present in an amount of about 0 to about 2.0 mM;HPO₄ ²⁻ is present in an amount of about 1.0 to about 10.0 mM; and HCO₃⁻ is present in an amount of about 5.0 to about 100.0 mM.
 12. A coatedsubstrate prepared by the method comprising: exposing a portion of asubstrate to an aqueous system at a temperature of about 20° C. to about80° C. to form a ceramic coating on a surface of the substrate; whereinthe aqueous system comprises a structural protein, a gelation inhibitoragent, a weak acid having a pKa of about 3.0 to about 5.5, water, Ca²⁺,HPO₄ ²⁻, a buffer system, and optionally one or more of Mg²⁺, Na⁺, K⁺,Cl⁻, SO₄ ²⁻; or HCO₃ ⁻; and wherein the aqueous system has an initial pHof about 5.0 to about 8.0.
 13. The coated substrate of claim 12, whereinthe structural protein is collagen Type I, II, III, or V.
 14. The coatedsubstrate of claim 12, wherein the gelation inhibitor agent is urea,histidine, hydroxyproline, thiourea, sodium dodecyl sulfate, lithiumdodecyl sulfate, 2-mercaptoethanol, formamide, dithiothreitol,(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate),(3-([cholamidopropyl]-dimethyl ammonio)-2-hydroxy-1-propanesulfonate),guanidinium chloride (guanidine hydrochloride), or guanidiniumthiocyanate.
 15. The coated substrate of claim 12, wherein the buffersystem comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid salts,tris-hydroxymethyl aminomethan, piperazine-1,4-bis(2-ethanesulfonicacid), piperazine-1,4-bis(2-ethanesulfonic acid) salts, or combinationsthereof.
 16. The coated substrate of claim 12, wherein the weak acid isan organic carboxylic acid having a pKa of about 3.0 to about 5.5. 17.The coated substrate of claim 12, wherein the structural protein iscollagen Type I present in an amount of about 0.1 g/L to about 5.0 g/L;the gelation inhibitor agent is urea present in an amount of about 0.1 Mto about 6.0 M; Ca²⁺ is present in an amount of about 2.5 to about 15.0mM; Mg²⁺ is present in an amount of about 0.5 to about 5.0 mM; Na⁺ ispresent in an amount of about 50.0 to about 300.0 mM; K⁺ is present inan amount of about 2.0 to about 20.0 mM; Cl⁻ is present in an amount ofabout 50.0 to about 350.0 mM; SO₄ ²⁻ is present in an amount of about 0to about 2.0 mM; HPO₄ ²⁻ is present in an amount of about 1.0 to about10.0 mM; and HCO₃ ⁻ is present in an amount of about 5.0 to about 100.0mM.
 18. The coated substrate of claim 12, wherein the exposing thesubstrate to the aqueous system occurs for a time of about 10 hours toabout 48 hours.
 19. The coated substrate of claim 12, wherein thesubstrate comprises a metal, a ceramic, a polymeric material, orsilicon.
 20. The coated substrate of claim 12, wherein the coating isperformed in a sealed container, wherein the sealed container comprisesa pressure valve.